Feasibility of biohydrogen production from cheese whey using a UASB reactor: Links between microbial community and reactor performance

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<ul><li><p>dun</p><p>community and reactor performance</p><p>E. Castello *, C. Garca y SanL. Borzacconia, C. EtchebehereaChemical Engineering Institute, School of EngbMicrobiology Department, School of Science a</p><p>production processes: electrolysis, natural gas reforming and</p><p>biological processes, such as dark fermentation of carbohy-</p><p>drate-rich substrates [1,2]. The main strategy for hydrogen</p><p>2</p><p>mol hexose1) [3]; however, the yields reported in the litera-</p><p>ture are lower [2,4]. This could be due to the incorporation of</p><p>substrate by the biomass, the production of fermentation</p><p>* Corresponding author. Facultad de Ingeniera, Universidad de la Republica, Herrera y Reisig 565, CP.11300, Montevideo, Uruguay.Tel.: 5982 711 08 71, 5982 711 44 78; fax: 5982 710 74 37.</p><p>Avai lab le at www.sc iencedi rect .com</p><p>w.</p><p>i n t e r n a t i on a l j o u r n a l o f h y d r o g e n en e r g y 3 4 ( 2 0 0 9 ) 5 6 7 4 5 6 8 2E-mail address: elenacas@fing.edu.uy (E. Castello).1. Introduction</p><p>Hydrogen is a clean energy carrier that possesses a high</p><p>energy yield (122 kJ g1) and does not contribute to thegreenhouse effect. There are many different hydrogen</p><p>production by dark fermentation is to block consumption by</p><p>methanogens and to select for high-yield hydrogen producers.</p><p>There are several genera of Bacteria known to produce</p><p>hydrogen by dark fermentation. Among them,members of the</p><p>Clostridium genera have the highest theoretical yield (4 mol H -Cheese whey fermentative organisms from the genera Megasphaera, Anaerotruncus, Pectinatus and Lacto-</p><p>bacillus, which may be responsible for hydrogen production. However, the persistence of</p><p>methanogenesis and the presence of other fermenters, not clearly recognised as hydrogen</p><p>producers indicates that competition for the substrate may explain the low hydrogen</p><p>production.</p><p> 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rightsreserved.a r t i c l e i n f o</p><p>Article history:</p><p>Received 9 March 2009</p><p>Received in revised form</p><p>7 May 2009</p><p>Accepted 14 May 2009</p><p>Available online 21 June 2009</p><p>Keywords:</p><p>Hydrogen production</p><p>UASB</p><p>Lactose fermentation0360-3199/$ see front matter 2009 Interndoi:10.1016/j.ijhydene.2009.05.060tos , T. Iglesias , G. Paolino , J. Wenzel ,b</p><p>ineering, University of the Republic, Herrera y Reissig 565, Montevideo, Uruguay</p><p>nd School of Chemistry, University of the Republic, General Flores 2124, Montevideo, Uruguay</p><p>a b s t r a c t</p><p>The present study examines the feasibility of producing hydrogen by dark fermentation</p><p>using unsterilised cheese whey in a UASB reactor. A lab-scale UASB reactor was operated</p><p>for more than 250 days and unsterilised whey was used as the feed. The evolution of the</p><p>microbial community was studied during reactor operation using molecular biology tools</p><p>(T-RFLP, 16S rRNA cloning library and FISH) and conventional microbiological techniques.</p><p>The results showed that hydrogen can be produced but in low amounts. For the highest</p><p>loading rate tested (20 gCOD/L.d), hydrogen production was 122 mLH2/L.d. Maintenance of</p><p>low pH (mean 5) was insufficient to control methanogenesis; methane was producedconcomitantly with hydrogen, suggesting that the methanogenic biomass adapted to the</p><p>low pH conditions. Increasing the loading rate to values of 2.5 gCOD/gVSS.d favoured</p><p>hydrogen production in the reactor. Microbiological studies showed the prevalence ofa, a b b bFeasibility of biohydrogen prowhey using a UASB reactor: Li</p><p>journa l homepage : wwational Association for Hction from cheeseks between microbial</p><p>e lsev ie r . com/ loca te /heydrogen Energy. Published by Elsevier Ltd. All rights reserved.</p></li><li><p>legislation that does not permit its land disposal without</p><p>i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 3 4 ( 2 0 0 9 ) 5 6 7 4 5 6 8 2 5675prior treatment, as well as economic reasons that force its</p><p>valorisation. Cheese whey has an elevated carbohydrate</p><p>(lactose) concentration and a low buffer capacity. Its treat-</p><p>ment in a conventional anaerobic reactor frequently leads to</p><p>acidification and inhibition of methanogenic activity [8].</p><p>These characteristics make this by-product a good substrate</p><p>for biohydrogen production. Hydrogen production should be</p><p>followed by a stage of methane production in order to</p><p>recover all the energy content of the cheese whey and</p><p>reduce the COD. There has been some experience working</p><p>with dry whey powder as a substrate for biohydrogen</p><p>production in continuous and batch modes [9,10], but the</p><p>ability to produce hydrogen using raw whey remains to be</p><p>evaluated.</p><p>Various technologies for hydrogen production can be</p><p>found in the literature, all of them at lab-scale: continuous</p><p>stirred tank reactors [9,11], sequencing batch reactors [12] and</p><p>upflow sludge bed reactors [13,14]. The operational conditions</p><p>that optimise the hydrogen production process have not been</p><p>completely defined, but pH and hydraulic retention time (HRT)</p><p>have been reported as the most important parameters to</p><p>control. To optimise the hydrogen production, reactors should</p><p>be operated at pH 5.5 with HRT between 8 and 12 h [2,1517];</p><p>however, there are also reports of a wider range of pH for</p><p>optimum operation, between 4.5 and 6.5 [6].</p><p>Most previous investigations were carried out using</p><p>synthetic wastewater. Therefore, more information is needed</p><p>on the applicability of the hydrogen production process to</p><p>industrial wastewater due to the possible presence of unde-</p><p>sirable microorganisms.</p><p>The objectives of this work were to study the applicability</p><p>of dark fermentation for hydrogen production in a UASB</p><p>reactor fed with raw, unsterilised cheese whey, and to eval-</p><p>uate the effect of increasing the organic loading rate. The</p><p>evolution of the microbial community was linked to the</p><p>reactor operational data to better understand the process.</p><p>2. Materials and methods</p><p>2.1. Substrate</p><p>Cheese whey was obtained from a local cheese production</p><p>factory. It was received from the factory once per week and</p><p>stored at 4 C until used. The average composition of thecheese whey was as follows: COD 67,000 mg/L (standard</p><p>deviation 6000 mg/L, 66 samples); total nitrogen 1335 mgN/L;products other than acetate and the consumption of hydrogen</p><p>by methanogens [2,5].</p><p>Biological hydrogen production has the advantage of</p><p>a low energy demand compared to other technologies [6].</p><p>The possibility of using organic wastes as substrates makes</p><p>the process even more attractive. Cheese whey is a by-</p><p>product generated during cheese manufacturing. The main</p><p>components are lactose (7072% dried extract), proteins (8</p><p>10%), and mineral salts (1215% of dried extract) [7]. Proper</p><p>management of cheese whey is important due to strictertotal phosphorus 310 mg/L; and pH 4.7 (standard deviation 0.9,</p><p>66 samples). Prior to being fed into the reactor, the whey wasdiluted to a COD concentration of 10,000 mg/L and supple-</p><p>mented with NaHCO3 (0.2 gNaHCO3/gCOD). The addition of</p><p>NaHCO3 was started at day 5 of operation after observing</p><p>a significant decrease in the pH of the reactor.</p><p>2.2. Seed sludge</p><p>The seed sludge was obtained from an acidogenic lab-scale</p><p>reactor fed with glucose that had been in operation for 3</p><p>months. No pre-treatment of the sludge was carried out prior</p><p>to its inoculation in the reactor.</p><p>2.3. The reactor system</p><p>A laboratory-scale UASB reactor (working volume 4.6 L, height</p><p>54 cm) with 4 sampling points along its height was used for</p><p>biohydrogen production. The reactor was placed in a 30 Cthermostatic chamber, and biogas production was measured</p><p>with a water displacement meter.</p><p>The reactor was started with a hydraulic residence time</p><p>(HRT) of 24 h and a COD concentration of 10,000 mg/L. The</p><p>COD level was kept constant during the operation. The</p><p>organic loading rate under those conditions was 10 gCOD/L.d.</p><p>To promote the elimination of methanogenesis, the HRT was</p><p>reduced to 12 h on day 56. From that point on, the organic</p><p>loading rate was 20 gCOD/L.d. Other changes in the hydraulic</p><p>residence time were due to operating problems.</p><p>2.4. Analytical methods</p><p>The determinations of chemical oxygen demand (COD), total</p><p>suspended solids (TSS) and volatile suspended solids (VSS)</p><p>were carried out according to standard methods [18].</p><p>Hydrogen and methane were determined by gas chromatog-</p><p>raphy (Chromatograph SRI 8610) using a molecular sieve 13column (Chrompack) and TCD detector. Volatile fatty acids</p><p>(VFA) were determined by HPLC with the following operating</p><p>conditions: polymeric column ORH-801, UV detector (Shi-</p><p>madzu 10AD) at 210 nm, mobile phase H2SO4 (0.005 M), a flow</p><p>rate of 0.8 mL/min, and an oven temperature of 45 C.</p><p>2.5. Microbiological studies</p><p>Samples (w25 mL) of suspended solids for microbiological</p><p>studies were taken during the operation of the reactor. Batch</p><p>tests and MPN culturing were performed immediately. For</p><p>fluorescence in situ hybridisation analysis (FISH) and DNA</p><p>extraction, the samples were centrifuged for 15 min at 6000 g</p><p>and 4 C. The supernatant fractions were decanted, and thecell pellets were stored at 20 C for DNA extraction or fixedwith paraformaldehyde [19] and then stored at 20 C forFISH.</p><p>The hydrogen production capacity was determined in</p><p>batch experiments measuring the specific hydrogen activity</p><p>as previously described [20]. The substrate was the same</p><p>waste product used for feeding the reactor in a final concen-</p><p>tration of 1000 mgCOD/L.</p><p>In specified samples (day 175 and 247), the presence of cellsfrom the domain Archaea were determined by FISH using the</p><p>Arc 915 probe [19] as previously described [21].</p></li><li><p>instructions. Colonies were chosen randomly, and sequences</p><p>i n t e r n a t i on a l j o u r n a l o f h y d r o g e n en e r g y 3 4 ( 2 0 0 9 ) 5 6 7 4 5 6 8 25676Samples taken on operation day 69 and 195 were used to</p><p>determine the number of hydrogen-producing bacteria by the</p><p>MPN method. Anaerobic medium supplemented with glucose</p><p>(Sigma, 10 g/L), triptone (Difco, 5 g/L), yeast extract (Difco, 5 g/</p><p>L) and meat extract (5 g/L) was used. The pH was adjusted to 5</p><p>with HCl, and bromocresol purple was used as the pH indi-</p><p>cator. The media was sparged in a N2 atmosphere and steri-</p><p>lised in an autoclave. After inoculation, tubes were incubated</p><p>at 30 C for 2476 h. Cultures were considered positive forhydrogen-producing bacteria when visible growth and</p><p>hydrogen in the gas phase (measured by GC) were present.</p><p>Positive cultures from the highest dilutions of the sample</p><p>taken at day 69were used to obtain isolates. Serial dilutions (1/</p><p>10) were performed in the same medium until only one</p><p>morphology was detected by microscopic observation using</p><p>Gram staining. Isolation was then performed in roll tubes</p><p>using the same anaerobic medium solidified with 2% agar</p><p>(Difco). Single colonies were picked and transferred to the</p><p>same liquid medium. The purity of the culture and the</p><p>morphology of the isolates were tested using Gram staining.</p><p>Isolates were characterised by 16S rRNA gene sequence anal-</p><p>ysis. DNA was extracted from liquid cultures using the DNA</p><p>Wizard extraction kit (Promega), carried out according to the</p><p>manufacturers suggestions for Gram positive bacteria. The</p><p>16S rRNA gene was amplified by PCR using the universal</p><p>Bacteria primers (forward 27F: 50-AGAGTTTGATCCTGGCTCAG-30, corresponding to positions 8 27 using Escherichia colinumbering; reverse1522R 50-AAGGAGGTGATCCAGCCGCA-30,corresponding to positions 1522 1542). Amplification reac-tions were performed as described in [22]. PCR product puri-</p><p>fication and sequencing were performed by Macrogene Inc.</p><p>(Korea) Sequencing service (Korea).</p><p>The utilisation of various substrates by the isolates was</p><p>determined using a basal anaerobic medium containing yeast</p><p>extract (Difco; 1 g/L) [22] supplemented with glucose (Sigma),</p><p>or sodium lactate (Aldrich) (10 mM). Growth was measured</p><p>spectrophotometrically (Genesys 5; Spectronic, Milton Roy) at</p><p>660 nm. Fermentation products (H2 and volatile fatty acids)</p><p>were determined for each different substrate as described.</p><p>Experiments were performed in triplicate.</p><p>The microbial community composition was studied</p><p>by Terminal Restriction Fragment Length Polymorphism</p><p>(T-RFLP) of the 16S rRNA present in the samples taken on the</p><p>following reactor operation days: 0, 69, 175 and 247. DNA was</p><p>extracted using an UltraClean Soil DNA Isolation Kit (MO BIO</p><p>Laboratories Inc.) according to the manufacturers protocol.</p><p>The 16S rRNA genes were amplified by PCR using the same</p><p>Bacteria universal primers used previously, but the forward</p><p>primer was fluorescently labelled with the dye 6-FAM (5-[6-</p><p>carboxy-fluorescein]). The amplification reaction was carried</p><p>out as described in [23]. The amplification products were</p><p>purified using a PCR purification kit (QIAGEN, Courtaboeuf,</p><p>France), and digested with the Msp I restriction enzyme (Fer-</p><p>mentas) according to the manufacturers suggestions. After</p><p>enzyme inactivation by heat treatment (65 C for 1 h), DNAfragments were precipitated with 90% ethanol and washed</p><p>twice in 70% ethanol. DNA fragments were dried at 65 C andthen re-suspended in 8 mL formamide and 0.3 mL of internalstandard (GeneScan-500 Liz Standard, Applied Biosystems).</p><p>The terminal restriction fragments (T-RF) were separated onfrom the plasmid insert were determined using the forward</p><p>primer of the cloned gene. DNA sequencing was conducted</p><p>using an ABI Prism 3700 gene analyzer (Applied Biosystems) at</p><p>the Michigan State University Genomics Technology and</p><p>Support Facility.</p><p>Sequences from clones and isolates were compared with</p><p>sequences from the NCBI database using Blastn Search (nucle-</p><p>otidenucleotide comparison) and from the Ribosomal Data-</p><p>base Project (RDP) using the Classifier Tool. Clones having a 16S</p><p>rRNA sequence similarity of more than 97% with each other</p><p>were grouped into an operational taxonomic unit (OTU).</p><p>Representative sequences were selected and aligned to related</p><p>sequences from the NCBI databasewith ClustalW, and a phylo-</p><p>genetic tree was constructed using MEGA version 3.1 [25]. Seq-</p><p>boot was used to obtain the confidence level in 500 datasets.</p><p>Sequences were digested in silico with the enzyme used</p><p>for T-RFLP to compare the T-RFLP peaks with the sequences</p><p>retrieved from strains and clones. The number of nucleotides</p><p>of the 50 in silico fragments were determined and comparedto the T-RF lengths.</p><p>3. Results and discussion</p><p>3.1. pH monitoring</p><p>After recording a significant drop in the reactor pH to 3.3</p><p>after 5 days of operation, the cheese whey feedstock was</p><p>supplemented with NaHCO3 to increase its alkalinity level</p><p>(0.2 gNaHCO3/gCOD). After pH recovery (around day 20), the</p><p>pH at the outlet was always greater than 4. The average pH at</p><p>the inlet and at the outlet of the reactor was 5 with a standard</p><p>deviation of 1 (49 samples). The observed pH variation at the</p><p>inlet over time may be explained by partial fermentation of</p><p>the whey, although it was maintained at 4 C until used.</p><p>3.2. Hydrogen production</p><p>The reactor operation started with a hydraulic residence timean ABI3130 Genetic Analyzer (Applied Biosystems) at the</p><p>Molecular Biology Unit (Institut Pasteur Montevideo). Chro-</p><p>matograms were analys...</p></li></ul>

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